Skeletal muscle is a unique organ that is composed of multinucleate muscle fibers, each of which is the product of fusion of hundreds or even thousands of myoblasts. Myoblast fusion is not only important for skeletal muscle development, but also critical for satellite cell-based muscle regeneration. Despite a large body of studies ove several decades, the mechanisms underlying myoblast fusion in humans remain poorly understood. Recent studies in the fruit fly Drosophila have begun to reveal unprecedented details about the molecular and cellular mechanisms of myoblast fusion. The striking evolutionary conservation between fly and mammalian myogenesis makes Drosophila a particularly relevant system to study myoblast fusion in vivo. Recent studies from our lab have uncovered a novel cellular mechanism underlying myoblast fusion. We show that myoblast fusion is mediated by a cell type-specific, F-actin-enriched podosome-like structure (PLS), which invades the opposing fusion partner with multiple protrusive fingers leading to fusion pore formation. To further characterize the regulation of the invasive PLS, the key cellular structure mediating myoblast fusion, we propose to investigate the molecular and cellular functions of two new genes, DPak3 and dynamin, both have previously been implicated in human diseases. We will use a multifaceted approach, including genetics, cell biology and biochemistry, to study the mechanisms by which DPak3 and dynamin regulate actin polymerization dynamics within the PLS in Drosophila myoblast fusion. We will also investigate the function of their mammalian orthologs in the fusion of mouse C2C12 myoblasts. Given the molecular and cellular conservation of myoblast fusion between fly and mammals, the proposed mechanistic studies using the simpler and genetically tractable Drosophila system will lead to significant insights int human muscle biology in health and disease, and ultimately provide a basis for developing more efficient therapeutics against the life debilitating muscle degeneration diseases. PUBLIC HEALTH RELEVANCE: Myoblast fusion is an essential step during skeletal muscle development and muscle stem cell-based regeneration. Understanding the mechanisms of myoblast fusion will lead to significant insights into human muscle biology in health and disease, and ultimately provide a basis for developing more efficient therapeutics against the life debilitating muscle degenerative diseases.